The present invention relates to the preparation and purification of envelope glycoproteins, particularly from the Paramyxoviridae family of human pathogenic viruses. The present invention also relates to the formulation of a mixture of the purified glycoproteins to give an efficacious and safe vaccine for use in infants and young children to protect against the diseases caused by the viruses.
Human Parainfluenza type 3 (PIV-3) and Respiratory Syncytial subgroups A and B (RSV-A,B) viruses, which are members of the Paramyxoviridae, have been identified as major pathogens responsible for severe respiratory disease in infants and young children. It has been shown that both formaldehyde-inactivated and live-attenuated vaccines failed to provide adequate protection against these diseases in clinical trials. Currently, safe and effective vaccines for prevention against these viral infections are not available. Thus, the development of efficacious PIV-3 and RSV vaccines is urgently required.
The major immunogenic proteins of RSV and PIV-3 have been identified, thereby providing the scientific rationale for a sub-unit approach to vaccine development. It has been shown that an in vivo protective response is contingent on the induction of neutralizing antibodies against the major viral surface glycoproteins. For PIV-3, these protective immunogens are the HN protein, which has a M.W. of 72 kDa, and possesses both hemagglutinin and neuraminidase activities and the F (fusion) protein, which has a M.W. of 65 kDa, and is responsible for both fusion of the virus to the host cell membrane and cell-to-cell spread of the virus. Immunogenicity studies in hamsters have shown that antibodies to both HN and F proteins were essential for protection against challenge with PIV-3. In addition, the presence of antibodies to both envelope glycoproteins was reported to correlate with protection in children naturally infected with PIV-3. For RSV, the two immunogenic surface glycoproteins are the 80-90 kDa glycoprotein (G) and the 70 kDa fusion (F) protein. The G and F proteins are thought to be functionally analogous to the PIV-3 HN and F proteins, respectively. In humans, antibodies to both PIV-3 viral surface glycoproteins are necessary for protection against PIV-3 infection, whereas anti-fusion protein antibodies are sufficient to elicit a cross-protective response against RSV infection.
In accordance with the present invention, the inventors have found a process for the production and purification of both PIV-3 and RSV viruses as well as a procedure for the purification of viral envelope surface glycoproteins generally. This process results in preparations that are potent PIV-3 immunogens in experimental animals and may be acceptable for use as vaccines in children. This procedure also is directly applicable for the production of virus and the purification of surface glycoproteins from any of the enveloped viruses, such as influenza, in which the major envelope proteins are important in eliciting an immunogenic response. The invention also includes the highly-purified immunogenic glycoproteins.
In the present invention, enveloped viruses, such as PIV-3 and RSV viruses, are grown in tissue culture on cell substrates that are readily acceptable for use in human vaccine production, such as the human diploid cell line MRC-5, in a medium virtually free of exogenous serum proteins. Surprisingly, under these conditions the cells continue to produce PIV-3 for more than three weeks, entirely in the absence of exogenously added growth factors. This process enables multiple virus harvests of similar antigenic composition to be obtained from the same group of cells. The absence of exogenous serum proteins greatly facilitates the process of purification.
Viral supernatants are either filtered or spun at low speed to remove cellular debris and concentrated by ultrafiltration, when necessary. The virus then is pelleted by ultracentrifugation. The virus also can be isolated by passage of the ultrafiltration retentate over an affinity matrix, such as Cellufine sulfate. The viral envelope glycoproteins then are solubilized with an appropriate detergent (eg. Triton X-100 or octylglucoside). Insoluble viral nucleocapsids are removed from the solubilized material by centrifugation. We have shown that this step, while useful, need not be performed. The viral surface glycoproteins are purified from the glycoprotein enriched fraction by affinity chromatography. Possible affinity matrixes include lentil-lectin and concanavalin A covalently coupled to cross-linked Sepharose or cellulosic microporous membranes. Contaminating cellular and residual viral matrix proteins are eliminated in the flow-through and high salt washes. Viral surface glycoproteins then are eluted from the column in the presence of an appropriate competing sugar, such as methyl xe2x88x9d-D-mannopyranoside, in the presence or absence of salt. Highly purified glycoprotein preparations (as judged by Coomasie blue or silver stained SDS polyacrylamide gels) are obtained using this process.
In accordance with the present process, HN and F from PIV-3 and F and G proteins from RSV were affinity-purified. The PIV-3 HN and F proteins were found to be highly immunogenic when tested in three separate animal models, namely guinea pigs, hamsters and cotton rats. Immunization of animals with varying doses of HN and F elicited a strong anti-glycoprotein antibody response. When administered with the appropriate adjuvants such as Freund""s or aluminum phosphate, the minimal immunoprotective dose can be significantly reduced. Thus the final vaccine preparation when formulated with aluminum phosphate as an adjuvant can be used as a readily injectable preparation for human use.
The effectiveness of the invention is not only limited to the preparation of the glycoproteins obtained from PIV-3 and RSV, but is applicable to coat proteins from all paramyxoviridae. Our invention also covers the use of similar methods of isolation and the use of adjuvants other than those mentioned.